1. Field of the Invention
The sporulating cells of Bacillus thuringiensis each produce a spore (endospore) and a diamond-shaped crystal (paraspore or inclusion body). At the completion of sporulation, the autolyzing cells release both of these bodies into the culture medium. The parasporal crystals are known to have insecticidal properties, and it is often desirable for investigative research and perhaps certain commercial purposes that these crystals be recovered in a substantially 100% pure state. However, it is difficult to separate the crystals from the spores because of similarity in their size and density. This invention relates to the removal of the interfering spores from a sporulated culture of B. thuringiensis in the course of isolating a pure crystal fraction.
2. Description of the Prior Art
Early techniques took advantage of the relative hydrophobicity of the spores as compared to the paraspores and isolated one from the other by phase separation. In the procedure described by T. A. Angus [J. Insect. Path. 1: 97-98 (1959)], the harvested bacterial culture was centrifuged repeatedly to remove the vegetative debris, and then stored for 7 days in a water suspension to germinate most of the spores. After four successive phase separations with a fluorocarbon, a crystal fraction of 95% purity was obtained. J. B. Bateson [Nature 205: 622-623 (1965)] was able to eliminate the need for germination and obtained crystals of 99% purity with only two successive fluorocarbon phase separations. However, the crystal yield was only about 3% and because of the high cost of the fluorocarbons, the procedure was not promising on a large scale. I. R. Pendleton et al. [Nature212: 728-729 (1966)] found that an inexpensive solvent, carbon tetrachloride, could be substituted for the fluorocarbon if 75% of the spores were first removed by repeated flotation steps. These were conducted by shaking the sporulated suspension until a froth formed on the surface, selectively entrapping a portion of the spores. The subsequent phase separation gave crystal purities of 98-99%, but the average yield was only 35%. R. E. Gingrich [J. Invertebr. Path. 10: 180-184 (1968)] improved somewhat upon the procedure of Pendleton by sparging a centrifuge supernatant with air bubbles in a column. The spore-laden bubbles were continuously removed, leaving the crystal-enriched tailings. While this process had certain potential for large-scale separation, it proved to be time-consuming and recovered only about 76% of the paraspores.
More recent methods of isolating crystals have included isopycnic centrifugation in gradients of cesium chloride [P. G. Fast, J. Invertebr. Path. 20: 139-140 (1972)], centrifugation in linear gradients of Renografin [E. S. Sharpe et al., Appl. Environ. Microbiol. 30: 1052-1053 (1975)], and centrifugation in discontinuous gradients of Renografin [R. Milne et al., J. Invertebr. Path. 29: 230-231 (1977)]. By these techniques, the vegetative cells and cellular debris are easily removed because of their low buoyant densities ranging from about 1.02 to 1.12 g./cm..sup.3. However, separation of the crystals from spores is not readily achieved due to the proximity of their respective buoyant densities, approximately 1.25 and 1.30 g./cm..sup.3. Overloading the gradient results in overlapping of the bands. This problem, together with the tendency of the hydrophobic spores to clump and entrap crystals, limits both the amount of mixture which can be separated at one time as well as the purity of the fractions.